Polyphenol Compositions

Abstract

The present application concerns polyphenol compositions, and the use of such compositions for preventing or treating endothelial dysfunction. The polyphenol compositions of the invention comprise at least one ellagitannin in combination with at least one proanthocyanidin.

Claims

1-36. (canceled)

37. A composition consisting essentially of at least one ellagitannin; at least one proanthocyanidin; and aspartic acid or an aspartate.

38. A composition consisting essentially of at least one ellagitannin; at least one proanthocyanidin; citrulline or a citrulline salt; and aspartic acid or an aspartate.

39. The composition of claim 37 or claim 38, wherein the at least one proanthocyanidin is at least one procyanidin.

40. The composition of claim 39 wherein the at least one proanthocyanidin is a mixture comprising procyanidins selected from the group consisting of procyanidin trimers, procyanidin tetramers, procyanidin pentamers and/or procyanidin hexamers.

41. The composition of claim 37 or claim 38, wherein the proanthocyanidin has a molecular weight of about 800 to about 1900 Daltons.

42. The composition of claim 37 or claim 38, wherein said proanthocyanidin is obtained from apple, cocoa bean, cranberry, grape seed, hawthorn, maritime pine bark or sorghum.

43. The composition of claim 42, wherein said proanthocyanidin is derived from grape seeds and is a galloylated procyanidin.

44. The composition of claim 37 or claim 38, where said ellagitannin has a molecular weight of about 1000 to about 3000 Daltons.

45. The composition of claim 37 or claim 38, wherein the source of said ellagitannin is selected from the group consisting of pomegranate fruits, raspberry fruits, and a plant in the Epilobium genus.

46. The composition of claim 45 wherein said plant is Epilobium parviflorum (smallflower hairy willowherb).

47. The composition of claim 37 or claim 38, wherein the at least one ellagitannin is oenothein B.

48. The composition of claim 37 or claim 38 wherein the weight ratio of the at least one ellagitannin to the at least one proanthocyanidin is in the range of about 10:1 to about 1:10.

49. The use of a composition of claim 37 or claim 38 as an ergogenic aid.

50. A method of preventing, delaying the onset of, or treating endothelial dysfunction comprising administering to a patient, either simultaneously or sequentially, oenothein B in combination with at least one proanthocyanidin, wherein said oenothein B and proanthocyanidin are administered in therapeutically effective amounts.

51. A method of preventing, delaying the onset of, or treating endothelial dysfunction comprising administering to a patient, either simultaneously or sequentially, at least one ellagitannin in combination with at least one proanthocyanidin, and aspartic acid or an aspartate, wherein said ellagitannin, proanthocyanidin, and aspartic acid or aspartate are administered in therapeutically effective amounts.

52. A method of preventing, delaying the onset of, or treating endothelial dysfunction comprising administering to a patient, either simultaneously or sequentially, at least one ellagitannin in combination with at least one proanthocyanidin, citrulline or a citrulline salt, and aspartic acid or an aspartate, wherein said ellagitannin, proanthocyanidin, citrulline or citrulline salt, and aspartic acid or aspartate are administered in therapeutically effective amounts.

53. The method of claim 51 or claim 52 wherein the ellagitannin is oenothein B.

54. The method of any one of claims 50 to 52 wherein the endothelial dysfunction is in the form of one or more diseases selected from arteriosclerosis, hypertension, pulmonary hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, diabetes, chronic renal failure and erectile dysfunction.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0136] FIG. 1: Biological activity of oenothein B and effect of oenothein B on binding of procyanidins to endothelial cells.

[0137] FIG. 2: Effect of oenothein B, raspberry seed ellagitannin extract, procyanidins and mixtures thereof on endothelin-1, a vasoconstrictor and biomarker associated with endothelial dysfunction.

[0138] FIG. 3: HPLC characterisation of oenothein B, and of procyanidin-enriched grape seed and apple extracts.

[0139] FIG. 4: Effect of oenothein B, procyanidins and mixtures thereof on biomarkers associated with reversal of endothelial dysfunction.

EXAMPLES

[0140] The following non-limiting Examples further illustrate the present invention.

[0141] For ease of reference, the following abbreviations are used herein:

A.sub.263—absorbance at 263 nm
ANOVA—analysis of variance statistical test
Ap-OPC—apple oligomeric procyanidins
Biotin-Ap-OPC—biotinylated apple oligomeric procyanidins
cm—centimetre
dP—degree of polymerisation (1=monomers to 10=decamers)
EllagiTs—ellagitannins
em—emission
eNOS—endothelial nitric oxide synthase
ET-1—endothelin-1
ex—excitation
g—gram
GS-OPC—grape seed oligomeric procyanidins
h—hour
HPLC—high performance liquid chromatography
kg—kilogram
KLF2—Kruppel-like factor 2
l litre
LC-MS/MS—liquid chromatography-tandem mass spectrometry
mg—milligram
μg—microgram
min—minute
ml—millilitre
mM—millimolar
μm—micrometer
μmol—micromolar
mRNA—messenger ribonucleic acid
mV—millivolts
NO—nitric oxide
nm—nanomolar
n.s.—non significant
OPC—oligomeric procyanidins
OTb—oenothein B
P—p-value, statistical significance
PTFE—polytetrafluoroethylene
qRT-PCR—quantitative reverse transcription polymerase chain reaction
Rb-ET—raspberry ellagitannins
SD—standard deviation
TMB—3,3′,5,5′-tetramethylbenzidine, chromogenic substrate
UHPLC—ultra high performance liquid chromatography
UV—ultraviolet

Example 1: Extraction and Partial Purification of Raspberry Ellagitannins

[0142] Aqueous acetone (70% in water), 3 l, is added to fresh seeds of red raspberries (approx. 2 kg) in a 5 l polypropylene beaker and mixed for 1 h at room temperature with a magnetic stirrer. The crude seed extract is filtered through a porous PTFE filter to separate the raspberry polyphenol extract (approx. 2.5 I) from particulate material. The resultant solution of raspberry polyphenols is diluted with 10 mM acetic acid (7.5 l) and pumped onto a column of Sephadex® LH20 (5×15 cm), which has been pre-equilibrated with 10% acetone in 10 mM acetic acid. The column is rinsed by pumping through 1 l of 10 mM acetic acid, followed by 1 l of 20% acetone in 10 mM acetic acid. Elution of raspberry ellagitannins is achieved by pumping 2 l 50% acetone in water. Fractions (200 ml) are collected. Fractions are monitored for polyphenol content by UV absorption measurements at 280 nm, and colorimetric assay measurements of polyphenols using Folin-Ciocalteu reagent (phosphomolybdate/phosphotungstate mixture). Fractions are also monitored for biological activity in relation to modifying endothelial function by assessing the relative potency of each fraction for inhibiting endothelin-1 synthesis by cultured endothelial cells.

[0143] The principles of this method can be adapted for pomegranate pericarp extraction. Aqueous acetone (70% in water), 600 ml, is added to finely chopped pomegranate pericarp (200 g). After filtration the filtrate (approx. 500 ml) is diluted with 10 mM acetic acid (1.5 l) and pumped onto a column of Sephadex® LH20 (5×15 cm). Subsequent steps are as described above.

Example 2: HPLC Purification of Fractions from Adsorption Chromatography

[0144] Peak fractions eluted with 50% aqueous acetone from Sephadex® LH20 are concentrated under vacuum to remove acetone. The concentrated extract is pumped onto a column of C18 silica (5×25 cm) at a flow rate of 50 ml/min. The column is rinsed with 1 litre of 8% methanol, and then eluted with 1.5 litre linear gradient to 25% methanol to obtain an enriched ellagitannin extract. 100 ml fractions are collected during elution. Fractions are monitored for polyphenol content by UV absorption measurements at 280 nm, and colorimetric assay measurements of polyphenols using Folin-Ciocalteu reagent (phosphomolybdate/phosphotungstate mixture). Fractions are also monitored for biological activity in relation to modifying endothelial function by assessing the relative potency of each fraction for inhibiting endothelin-1 synthesis by cultured endothelial cells. Peak fractions, based on ability to inhibit endothelin-1 synthesis, are pooled and dried.

Example 3: Purification of Apple Procyanidins

[0145] 20 g of crude apple polyphenol extract powder (75% polyphenol extract from A. M. Todd Botanical Therapeutics, Kalamazoo, Mich., USA) is dissolved in 500 ml 10% acetone in 10 mM acetic acid. The crude extract is pumped onto a column of Sephadex LH20 (5×15 cm). Subsequent steps are as described above. The column is then rinsed and eluted with 50% aqueous acetone as described in Example 1. The yield of procyanidins is 7 g (35%). Relative potency of the purified extract compared to the starting material is determined by comparing inhibitory effects on endothelin-1 synthesis by endothelial cells. The proportion of procyanidin tetramers to hexamers in the purified extract, compared to the starting material, is assessed by HPLC using a Develosil® 100 Diol column (5 μm, 4.6×250 mm, available from www.develosil.com). Such methods are well known in the art, see e.g. Journal of Agricultural and Food Chemistry 2009, 57: 1896-902 and Journal of Chromatography A, 2009, 1216, 4831-4840. The purified apple procyanidins are pooled and dried.

Example 4: HPLC and LC-MS/MS Analysis of Polyphenols

[0146] The procyanidin content of extracts is analysed by HPLC using a Develosil 100 Diol column (5 μm, 4.6×250 mm) with procyanidins monitored by fluorescence (ex 276 nm, em 316 nm) using gradient elution with acetonitrile/acetic acid/methanol/water. The identities of the eluted procyanidins can be further confirmed by connecting this HPLC system to a mass spectrometer for MS/MS analyses.

[0147] Ellagitannin compositions of extracts are analysed by reverse phase HPLC using a UHPLC column of C18 silica (2×50 mm) connected to a mass spectrometer for MS/MS analyses (as per the methods described in Molecules, 2012, 17:14821-40).

Example 5: Inhibition of Endothelin-1 Synthesis by Endothelial Cells

[0148] The effects on endothelin-1 synthesis of purified procyanidins and ellagitannins, either alone or in combination, are tested on human, bovine or porcine aortic endothelial cells cultured in multi-well plates. Cells are grown in a CO.sub.2 incubator with specific growth media until becoming confluent. Purified extracts, chromatography fractions, or isolated procyanidins and ellagitannins are evaluated after dilution in cell culture media and then incubated for up to 24 h with the cultured cells. At the end of the incubation period cell culture media samples are collected, and endothelin-1 synthesis is determined by immunoassay of the media samples. ET-1 release for each treated well is expressed as a percentage of basal release from control wells containing cells incubated with cell culture medium alone. Data are compared and analysed for statistically significant differences using life sciences software such as Graphpad Prism (available from www.graphpad.com). At the end of the incubation period cells are lysed to obtain mRNA to measure changes in gene expression that indicate improved endothelial function. Analyses of relative levels of mRNA transcripts are undertaken by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Journal of Agricultural and Food Chemistry, 2010, 58: 4008-13).

Example 6: Stimulation of Nitric Oxide Synthesis by Endothelial Cells

[0149] Confluent cultures of endothelial cells are prepared as described in example 5. Acute changes in nitric oxide synthesis can be stimulated with the calcium ionophore A23187 (0.1-1 μM), after cells have been preincubated for up to 24 h with purified procyanidins and ellagitannins, either alone or in combination. The influence of citrulline and aspartate on NO synthesis is evaluated by adding citrulline (0.05-1 mM) alone or in combination with aspartic acid (0.05-1 mM) to the cell culture medium during periods when NO measurements are made. NO measurements are made by assaying total nitrite and nitrate in conditioned media samples using the Griess reaction, monitored spectrophotometrically at 540 nm; or by measurement of NO by fluorescence with DAF-2 (4,5-diaminofluorescein) (FEBS Letters 1998, 427: 263-266). At the end of incubations cells can be lysed for measurement of eNOS protein by western blot as index of eNOS activity.

Example 7: Endothelium-Dependent Vasodilatation of Blood Vessels

[0150] Isolated blood vessels sections are prepared from rat aorta and cut into rings, carefully avoiding damage to the endothelium. Vascular rings are individually suspended in organ baths and bathed in Krebs-Ringer solution. Vascular rings are attached to isometric transducers to measure vascular tone. After a period of equilibration the rings of aorta are pre-contracted with phenylephrine (1 μmol/l) and the presence of a functional endothelium is verified with acetylcholine (10 μmol/l). Relative effectiveness of procyanidins and ellagitannins as inducers of endothelium-dependent vasodilatation is assessed by constructing cumulative concentration response curves with purified procyanidins and ellagitannins, either alone or in combination. Citrulline (0.05-1 mM) is added alone or in combination with aspartic acid (0.05-1 mM) to the organ bath solution prior to addition of procyanidin/ellagitannin combinations and compared to the same concentrations of procyanidin and ellagitannin without citrulline and aspartic acid. Data are compared and analysed for statistically significant differences using life sciences software such as Graphpad Prism.

Example 8: Pharmaceutical Product for Treatment of Chronic Heart Failure

[0151] A tablet or capsule pharmaceutical formulation is made containing 125 mg purified raspberry ellagitannins as per Example 2 and 125 mg purified apple procyanidins as per Example 3, plus appropriate pharmaceutical excipients. Two to eight tablets are to be taken per day. Biomarker monitoring of the response to treatment may be made by measurements of N-terminal pro-B-type natriuretic peptide at monthly intervals.

Example 9: Pharmaceutical Product for Treatment of Chronic Heart Failure

[0152] A tablet or capsule pharmaceutical formulation is made containing 125 mg purified Epilobium ellagitannins including oenothein B and 125 mg purified apple procyanidins as per Example 3, plus appropriate pharmaceutical excipients. Two to eight tablets are to be taken per day. Biomarker monitoring of the response to treatment may be made by measurements of N-terminal pro-B-type natriuretic peptide at monthly intervals.

Example 10: Microencapsulation of Procyanidins and Ellagitannins

[0153] To increase stability, mask flavour and increase bioavailabilty, ellagitannins and procyanidins are microencaspulated using water-in-oil microencapsulation technology for liquid products; or maltodextrin, mesquite gum with zein and spray dried for a powder product. These microencapsulated polyphenols can be incorporated directly into liquid products such as in Examples 11 and 12, or the spray dried microencapsulated polyphenols can be incorporated into dry powder products such as in Examples 13 and 14.

Example 11: Drink Product for Treatment of Hypertension

[0154] 250 mg raspberry ellagitannins obtained by the method of Example 2 and 250 mg apple procyanidins obtained by the method of Example 3 are microencapsulated. To this mixture is added 4 g citrulline and 3 g aspartic acid. The mixture is diluted with 50 ml of water containing pH stabilisers, sweeteners and flavourings. One to two drinks of 50 ml may be consumed per day.

Example 12: Drink Product for Treatment of Hypertension

[0155] 250 mg ellagitannins obtained from pomegranate (including oenothein B) and 250 mg procyanidins obtained from grape seed extract are microencapsulated. To this mixture is added 4 g citrulline and 3 g aspartic acid. For use, the mixture is diluted with 50 ml of water containing pH stabilisers, sweeteners and flavourings. One to two drinks of 50 ml may be consumed per day.

Example 13: Ergogenic Product for Improving Athletic Performance and Duration of Maximum Performance

[0156] Dried powder in a sachet containing lyophilised microencapsulated 500 mg raspberry ellagitannins and 500 mg apple procyanidins obtained via the methods described in Examples 2 and 3 are mixed in powder form with citrulline 5 g, aspartic acid 4 g, soya protein hydrolysate powder 25 g, L-carnitine tartrate 2 g, ascorbic acid 30 mg, tocopherol acetate 5 mg, and appropriate flavourings and/or sweetener as required.

[0157] For use, the full contents of a sachet are mixed with 300 ml water. One to two sachets may be consumed per day.

Example 14: Ergogenic Product for Improving Athletic Performance and Duration of Maximum Performance

[0158] Dried powder in a sachet containing lyophilised microencapsulated 500 mg ellagitannins obtained from pomegranate (including oenothein B) and 500 mg procyanidins obtained from grape seed extract are mixed in powder form with citrulline 5 g, aspartic acid 4 g, soya protein hydrolysate powder 25 g, L-carnitine tartrate 2 g, ascorbic acid 30 mg, tocopherol acetate 5 mg, and appropriate flavourings and/or sweetener as required.

[0159] For use, the full contents of a sachet are mixed with 300 ml water. One to two sachets may be consumed per day.

Example 15: Inhibition of Endothelin-1 Synthesis by Oenothein B

[0160] Oenothein B (>95% purity) was purified from pomegranate juice by Sephadex LH20 chromatography and reverse-phase HPLC (step 1—Apex Prepsil 8 μm C-18 10×250 mm HPLC column—Jones Chromatography; step 2 Luna 5 μm PFP(2) 4.6×250 mm HPLC column—Phenomenex). Ability to modify endothelial function was assessed by incubating oenothein B with bovine aortic endothelial cells and measuring endothelin-1 synthesis following previously described methods (Journal of Agricultural and Food Chemistry, 2010, 58: 4008-13). As shown by FIG. 1(A), inhibition of endothelin-1 synthesis by oenothein B was found to be concentration dependent.

Example 16: Inhibition of Endothelin-1 Synthesis by Biotinylated Apple Oligomeric Procyanidins (Biotin-OPC)

[0161] Biologically active biotin-labelled apple oligomeric procyanidins were prepared as follows: apple polyphenol extract was fractionated on Sephadex LH20 to obtain a fraction enriched in apple oligomeric procyanidins (OPC) tetramers to hexamers. The fraction was dried, re-dissolved in dimethyl formamide and reacted with the tetrafluorophenyl ester of triethyleneglycol-biotin (Thermo Scientific EZ-Link™ TFP-PEG3-Biotin). The resultant biotinylated-OPC product was purified by HPLC and retention of biological activity was confirmed using bovine aortic endothelial cells by measuring inhibition of endothelin-1 synthesis. As shown by FIG. 1(B), inhibition of endothelin-1 synthesis by biotinylated apple oligomeric procyanidins was observed.

Example 17: Increased Binding of Biotinylated OPC to Endothelial Cells

[0162] Bovine aortic endothelial cells were incubated with oenothein B (μg/ml) from pomegranate juice for 30 min before addition of biotinylated-OPC (1 μg/ml) alone or with oenothein B (μg/ml) for 30 min. After removal of the incubation mixture, bound biotinylated-OPC was detected by incubation with neutravidin-horseradish peroxidase for 1 h; followed by addition of TMB substrate for 30 min after cells had been rinsed twice to remove excess neutravidin-horseradish peroxidase. Binding is indicated relative to biotinylated-OPC alone after subtraction of background signal. (All data are shown as mean±SD; *=P<0.05; **=P<0.01; ***=P<0.001 indicates significant differences from basal or control values). As shown by FIG. 1(C), binding of procyanidins increases as oenothein B concentration increases. From this it can be concluded that oenothein B increases the affinity of endothelial cells for procyanidins, and enhances the effectiveness of procyanidins for reversing endothelial dysfunction.

Example 18: Effect of Oenothein B, Ellagitannin Extracts, Procyanidins and Mixtures Thereof on Endothelial Cell Synthesis of Endothelin-1

[0163] Oenothein B (OTb, >90% purity) was purified from extracts of Epilobium by Sephadex LH20 chromatography and reverse phase C-18 HPLC (Apex Prepsil 8 μm C-18 10×250 mm HPLC column—Jones Chromatography). Analysis of purity was performed by HPLC using a Luna 5 μm PFP(2) 4.6×250 mm HPLC column (Phenomenex) by gradient elution at 1 ml/min with methanol (0-10% over 2 min, 10-25% over 30 min, and 25-80% over 10 min) in 10 mM acetic acid, UV absorbance was measured at 263 nm, FIG. 3(A). Raspberry ellagitannin extract (Rsb) was prepared from raspberry seeds (see EXAMPLE 1). Grape seed extract procyanidins were prepared from MegaNatural Gold grape seed extract (Polyphenolics Inc, Madera, Calif., USA), by fractionation on Sephadex LH20, with the enriched fraction of grape seed procyanidins (GS-OPC) being eluted with 50% acetone. The composition of GS-OPC was determined by HPLC using a 100Diol-5 Develosil™ HPLC column (Nomura Chemical Co. Ltd, Japan) with gradient elution at 0.8 ml/min: the column was equilibrated with 95% buffer A/5% buffer B and eluted with a gradient to 40% buffer B over 35 min and maintained at 40% buffer B for 10 min (buffer A—98% acetonitrile/2% acetic acid; buffer B—95% methanol/3% water/2% acetic acid) and monitored by fluorescence Ex 276 nm/Em 336 nm). The composition of GS-OPC was enriched in oligomeric procyanidins in the trimer to hexamer range (OPC trimer to hexamer >50%; detailed composition: monomer 3%, dimer 18%, trimer 16%, tetramer 16%, pentamer 12%, hexamer 8% heptamer 6%, unresolved OPC ≈20%) FIG. 3(B). Apple OPC were fractionated on Sephadex LH20 to enrich the composition of oligomeric procyanidins in the trimer to hexamer range (OPC trimer to hexamer 70.6%; detailed composition: monomer 1.6%, dimer 18.0%, trimer 24.9%, tetramer 20.4%, pentamer 15.2%, hexamer 10.1% heptamer 5.6%, octamer 2.8%, nonamer 1.1%, decamer 0.3%) FIG. 3(C); analysis was undertaken using a 100Diol-5 Develosil™ HPLC column (Nomura Chemical Co. Ltd, Japan) as for GS-OPC.

[0164] Ellagitannins: oenothein B (OTb, 1 μg/ml), raspberry seed extract (Rb-ET 2 μg/ml); oligomeric procyanidins (OPC): grape seed extract (GS-OPC, 1 μg/ml) and apple extract (Ap-OPC, 1 μg/ml) purified as above, were incubated either alone, or in combination, with bovine aortic endothelial cells for 6 h and 24 h (FIGS. 2(A) and 2(B)). After the respective incubation periods, conditioned media samples were collected for assay of endothelin-1 (see Journal of Agricultural and Food Chemistry, 2010, 58: 4008-13). Data (mean±SD) are shown as a percentage of the corresponding basal values at 6 h or 24 h. Statistical differences were determined by ANOVA (**=P<0.01; ***=P<0.001 indicates significant difference compared to basal values; .sup.+++=P<0.001 compared to the individual components of the combinations alone).

[0165] While the ellagitannins (oenothein B, raspberry seed extract) and oligomeric procyanidins (grape seed extract, GS-OPC, and apple extract, Ap-OPC) alone all inhibited endothelin-1 synthesis over 6 h the combinations had a greater effect than the extracts used separately (FIG. 2(A)). However, after 24 h the effects of the extracts alone were much reduced with only raspberry seed extract and apple extract causing a significant reduction. In comparison the combinations of ellagitannins and procyanidins caused a remarkably sustained and increased reduction in endothelin-1 synthesis (FIG. 2(B)).

[0166] These data clearly show that use of an ellagitannin in combination with a procyanidin produces a synergistic effect on endothelial function that has a much greater magnitude of response and longer duration of action than either ellagitannins or procyanidins alone. The consequence of this combination is likely to be greater and longer-lasting reversal of endothelial dysfunction.

Example 19: Effect of Oenothein B, Procyanidins and Mixtures Thereof on Biomarkers Indicating Reversal of Endothelial Dysfunction

[0167] Oenothein B (OTb, 1 μg/ml) and apple oligomeric procyanidins (Ap-OPC, 1 μg/ml) purified as above (see EXAMPLE 18), were incubated either alone, or in combination, with bovine aortic endothelial cells for 24 h. After 24 h incubation, conditioned media samples were collected for assay of endothelin-1, and cells were lysed for measurement of relative levels of mRNA for endothelin-1, Kruppel-like factor 2 (KLF2) and endothelial nitric oxide synthase (eNOS) by qRT-PCR (see Journal of Agricultural and Food Chemistry, 2010, 58: 4008-13). Levels of mRNAs were normalised to the mRNA levels of the housekeeping gene RNA polymerase 2, and are shown in FIG. 4 as ratios to control levels for the respective mRNAs. Data are shown as mean±SD. Statistical differences were determined by ANOVA (**=P<0.01; ***=P<0.001 indicates significant difference between the effects of OTB and OPC in combination compared to control values, oenothein B alone and apple OPC alone).

[0168] While use of either oenothein B or apple OPC alone for 24 h has some effect on KLF2 mRNA levels, the effects are relatively insignificant (see FIG. 4(A)). However, when oenothein B is used in combination with apple OPC, the increase in KLF2 mRNA levels is remarkable, and is indicative of a synergistic interaction between the ellagitannin and procyanidin. Similar results are found on other biomarkers of endothelial function. Thus, use of either oenothein B or apple OPC has little effect on eNOS mRNA levels after 24 h (see FIG. 4(B)). However, when oenothein B and apple OPC are used in combination, a significant increase in eNOS expression is observed. Turning to FIGS. 4(C) and 4(D), endothelin-1 (ET-1) mRNA and endothelin-1 total synthesis is largely unaffected by use of either oenothein B or apple OPC alone for 24 h, while use of oenothein B in combination with apple OPC results in a marked decrease in endothelin-1 mRNA levels and total endothelin-1 synthesis.

[0169] These data clearly show that use of an ellagitannin in combination with a procyanidin/proanthocyanidin can produce a synergistic effect on functional biomarkers that indicate reversal of endothelial dysfunction.